Project Summary/Abstract: Our recent work showed that triggering the anti-oxidant master regulator Nrf2, in macrophages, establishes an antiviral state in those cells that blocks HIV-1 and HIV-2 infection as well as infection by animal viruses FIV and MVV. Investigating the impact of the block on HIV-1, we found that it is manifest after reverse transcription, likely before the viral DNA enters the cell nucleus. Nrf2 regulates upward of one hundred genes. Our work to determine which of these blocks infection revealed that one, a cell surface protein that regulates the intracellular oxidative state by importing cystine, also contributes to determining the infectability of macrophages. Interestingly, we found that this protein and the block are already partially active in macrophages, thus allowing both up-and down-regulate the antiviral effects. How this cell surface protein blocks a virus infection step within the cell is not known. The long-term goal of our work is to better understand cellular defenses against viral infection so that they can be controlled therapeutically to benefit the host. The overall objective of the work proposed in this application is to determine whether xCT acts alone to block HIV or whether it is a part of a more complex anti-viral pathway. Our central hypothesis is that xCT blocks nuclear import of the viral pre-integration complex by acting directly on the virus, perhaps by trapping it at the cytoplasmic membrane after entry. The rationale for the proposed research is that understanding the role of xCT in the Nrf2-regulated restriction will direct our subsequent research into how the block works. Knowing how xCT contributes to hindering retrovirus infection will help to reveal how this mechanism can be exploited in either myeloid-specific or universal antiviral strategies. Our hypothesis will be tested in three specific aims: (1) To determine whether xCT alone is sufficient to block macrophage infection, (2) To test whether the amino acid transporter function of xCT is required for the Nrf2-triggered infection block and (3) To identify where in the cell the viral components are blocked. Since it?s not clear how a cell surface protein acts to cause an apparent nuclear import block, our approach is to test whether xCT acts directly, or indirectly through its normal function, and where in the cell this impacts the virus. We believe that the work proposed in this application is innovative because it will offer the opportunity to refocus the study of host-antiviral factors to include a new, exogenously inducible, restriction pathway. This contribution will be significant because it is expected to offer a new pathway for retrovirus control that can be externally up- or down-regulated in macrophages and possibly in related cell types like DCs and microglia.